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Biomedical subjects

J R Garrett

Publications and source records attributed to J R Garrett.

At least 19 recordsLinked to original sources
PubMed1992

New specific assays for tonin and tissue kallikrein activities in rat submandibular glands. Assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva.

At least fourteen separate bands of proteinase activity, labelled A-N, were identified by an enzyme overlay membrane technique, using oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) substrates in rat submandibular gland extracts fractionated on pH 4-6.5 isoelectric focusing gels. The proteinases were eluted into an ammonium bicarbonate buffer pH 9.8 containing 0.1% Triton X-100 and the relative contribution of each band to total activity evaluated using D-Val-Leu-Arg-AFC (DVLR-AFC) and Z-Val-Lys-Lys-Arg-AFC (ZVKKR-AFC) as substrates. Immunoblotting of band eluants run on sodium dodecyl sulphate gels with antibodies showed that band A was identical with tonin and bands K-N contained tissue kallikrein. Tonin was found to hydrolyse ZVKKR-AFC but not DVLR-AFC. Estimates of the Km values of tissue kallikrein for DVLR-AFC and tonin for ZVKKR-AFC were found to be similar (approx. 20 microM) yet the former enzyme hydrolysed its substrate five times faster. Tonin was inhibited by soybean trypsin inhibitor (SBTI) but not by aprotinin. Tissue kallikrein, on the other hand, was inhibited by aprotinin but was considerably more resistant to inhibition by SBTI. In tissue extracts 95% of the ZVKKR-AFC lytic activity in the presence of 1 microM aprotinin is due to tonin and a similar percentage of the DVLR-AFC hydrolysing activity in the presence of 10 microM SBTI is due to tissue kallikrein. These findings were used for the specific measurement of these two proteinases in submandibular gland extracts and in saliva without prior purification. Using these inhibitor based assays we revealed qualitative differences in the composition of proteinases secreted into saliva during parasympathetic versus sympathetic stimulation of the submandibular gland. The distribution of proteinases in sympathetic saliva is very similar to that found in submandibular extracts but on parasympathetic stimulation, although much less proteinase is released, the contributions of the more acidic isomers of tissue kallikrein are increased and that of tonin and other proteinases dramatically decreased. The data suggest that parasympathetic and sympathetic nerves induce proteinase secretion via different pathways.

Amino Acid Sequence↗
PubMed1992

Na,K-ATPase in resting and stimulated submandibular salivary glands in cats, studied by means of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity.

Sections from normal resting submandibular glands were incubated in a p-nitrophenylphosphate substrate mixture to demonstrate Na,K-ATPase activity by light and electron microscopy. Dense deposits of reaction product occurred within the basal and lateral infoldings of striated duct cells. A similar, less heavy deposition occurred in demilune cells. Only scattered, infrequent pockets of staining occurred in association with the basal plasma membrane of central acinar cells, supporting the belief that these cells do not contribute greatly to the secretion of fluid. No reaction was detected on luminal membranes. Glands tested after sympathetic or parasympathetic nerve stimulation showed no obvious differences from resting glands. These results contrast with the previously reported paucity of immunostaining in resting demilunes with an antibody to Na,K-ATPase and the large increase in the immunostaining by these cells after parasympathetic stimulation. It is inferred that there must be an inaccessibility to immunobinding sites of Na,K-ATPase in resting glands, and that the increase in binding which occurs after parasympathetic stimulation represents a conformational change that did not affect its enzyme catalytic reaction with this substrate.

4-Nitrophenylphosphatase↗
PubMed1992

Secretory oedema in diabetic submandibular glands during parasympathetic nerve stimulation: relationship to microvascular abnormalities in streptozotocin-treated rats.

1. Submandibular secretion during parasympathetic stimulation (5 Hz) was examined in streptozotocin-diabetic and age-matched control rats. 2. At 3 weeks, but not 3 and 6 months, flow rate was initially greater than in controls, but it declined rapidly after 30 min. 3. The reduction in flow rate was associated with oedema of the gland. 4. At 3 months, graded stimulation revealed a tendency to oedema at frequencies of 10 Hz and above. 5. Morphologically, submandibular capillary density was increased in diabetic rats. 6. Thus, in diabetes the submandibular gland appears less able to withstand continuous parasympathetic stimulation, due in part to an increase in tissue capillary area.

Animals↗
PubMed1992

Endothelium-derived vasodilator responses to sympathetic stimulation of the submandibular gland in the cat.

1. The extent to which vasodilator responses to electrical stimulation of the sympathetic innervation, in the submandibular gland of the cat, depend upon release of endothelium-derived relaxing factor (EDRF) within the gland has been investigated in anaesthetized cats given N-nitro-L-arginine methyl ester (L-NAME) which specifically blocks the synthesis of EDRF from arginine. 2. Close intra-arterial infusions of L-NAME (> or = 100 mg kg-1) produced a steady and significant rise in mean aortic pressure together with a steady increase in basal submandibular vascular resistance over the next 20-30 min. It also reduced, but failed to abolish, the vasodilatation which occurs during intermittent stimulation of the sympathetic innervation (20 Hz for 1 s at 10 s intervals) together with the after-dilatation which occurs immediately after a period of continuous stimulation of these nerve fibres (2 Hz). 3. In cats pretreated with the beta-blocker propranolol (> or = 1.0 mg kg-1) both vasodilator responses were reduced, but persisted until L-NAME was administered, whereupon both were abolished. 4. It is concluded that release of EDRF within the submandibular gland of the cat contributes to the basal tone of the vasculature and is responsible for the alpha-adrenergic vasodilator responses to stimulation of the sympathetic innervation, but not for the beta-adrenergic vasodilator responses.

Animals↗
PubMed1979

New observations on the innervation of striated ducts in submandibular glands of cats, including possible peptidergic nerves.

The presence of hypolemmal axons between striated duct cells in submandibular glands of cats has been established electron microscopically. Axons were found between "light" cells, between "light" and "dark" cells and between "light" and basal cells. Hypolemmal axons were observed most frequently in the junctional region between striated and intercalary ducts. They were often more common in younger animals. "Dark" cells with numerous processes sometimes appeared to have a special relationship with hypolemmal axons. Most of the hypolemmal axons in striated ducts contained characteristic agranular vesicles of the cholinergic type, about 40 nm in diameter; many of these axons also contained large dense cored vesicles of the peptidergic type, about 100 nm in diameter and possessing a more clear outer halo. No adrenergic axons have been observed beneath the basal lamina of striated ducts, even after use of 5-OHDA. The possibility that some of the hypolemmal axons in striated ducts are peptidergic and their possible functions are discussed. Apart from other activities these axons may have a role in supplying special trophic factors to the cells, helping them in their developmental specialisation and maintaining them in normal condition. An absence of such factors after parasympathetic decentralisation may be responsible for the dramatic atrophic changes in striated duct cells, especially since the atrophy in the gland is not solely due to an absence of acetylcholine activation.

Animals↗
PubMed1979

Activities of salivary myoepithelial cells: a review.

Historical developments concerning salivary myoepithelial cells have been outlined and structural features of the cells have been described. Evidence from structural and functional studies supports the belief that myoepithelial cells usually have a dual innervation by parasympathetic as well as sympathetic nerves, and impulses from both types of nerve cause the cells to contract. Functional assessment using salivary flow phenomena and intraluminal pressure changes have been used to determine, so far as is possible, the effects of myoepithelial contractions. In some instances tissues have been examined structurally after such experimental procedures. These investigations indicate that salivary myoepithelial activity 1. Speeds up the outflow of saliva 2. Reduces luminal volume 3. Contributes to the secretory pressure 4. Supports the underlying parenchyma 5. Helps salivary flow to overcome increases in peripheral resistance. However, beyond a certain point, this may lead to sialectatic damage of striated ducts with increase in glandular permeability. Myoepithelial activity may also help to expel parenchymal cell contents in certain instances.

Adrenergic Fibers↗
PubMed1979

Changes in the main submandibular salivary duct of rabbits resulting from ductal ligation.

Normal submandibular ducts from rabbits have been examined by mucosubstance histochemistry, transmission and scanning electron microscopy. The results were compared with the appearances of ducts removed 4...6 weeks after ligation. The normal ducts were composed mainly of columnar "light" cells and basal cells but, in addition, some "dark" cells and scattered goblet containing sulphated mucins were always present. The luminal surface of the ductal cells possessed numerous microvilli protruding into the lumen, and a rim of negatively charged mucin was present on this surface of these cells. After ligation the ducts became greatly distended by their fluid contents which remained under pressure until the duct was incised. The epithelial cells were flattened and appeared to contain less cytoplasm per cell; "light" cells, basal cells and "dark" cells were still recognisable. Goblet cells were much more plentiful than in the control ducts and often protruded into the lumen despite the increased intraluminal pressure. The development of a number of ciliated cells had also occurred and they were often situated close to goblet cells. Lymphatic vessels were more prominent around the ligated ducts. Luminal microvilli were less numerous than in the control ducts but the rim of negatively charged mucin on the luminal surface of ductal cells was more conspicuous. Mixed inflammatory cells were present within the lumina of ligated ducts especially in those parts adjacent to the ductal cells. No inflammatory cell has been observed passing through the wall of a main duct and the possibility exists that these cells had entered lumina within the gland and migrated from there to the main duct. The above findings may serve to help our understanding or physiological events in the ducts.

Animals↗
PubMed1977

The autonomic innervation of rabbit salivary glands studied electron microscopically after 5-hydroxydopamine administration.

In submandibular glands of rabbits both adrenergic and cholinergic axons are intimately associated with parenchymal cells of the intercalary ducts and the granular tubules, lying beneath the basement membrane and often in the space between the parenchymal cell and an associated myoepithelial cell. The submandibular acini receive a less intimate and less plentiful innervation by adrenergic and cholinergic axons which remain outside the basement membrane and are still associated with Schwann cells. Occasional axons of both adrenergic and cholinergic type occur beneath the basement membrane of submandibular striated ducts in intimate association with basal parts of the cells. In the parotid glands numerous adrenergic and cholinergic axons are found beneath the basement membrane of acini and intercalary ducts in intimate association with the cells.

Adrenergic Fibers↗
PubMed1977

Adrenergic influences on the permeability of rabbit submandibular salivary glands to blood-borne horseradish peroxidase.

Horseradish peroxidase (HRP) administered close-arterially, has been found to enter rabbit submandibular saliva elicited by parasympathetic nerve stimulation. Adrenalin, superimposed on parasympathetic nerve stimulation, increased the passage of HRP into the saliva. Use of alpha- and beta-adrenoceptor agonists, either separately or together, and use of alpha- or beta-adrenoceptor antagonists together with adrenalin indicate that both alpha- and beta-receptor stimulation is necessary for this increase in glandular permeability to occur. Histochemical assessment showed that HRP had permeated the interstitial spaces of the gland and entered the spaces between adjacent parenchymal cells. However, in unstimulated glands it had only reached the lumina of striated ducts, but after adrenalin administration, peroxidase was also observed within acinar lumina. This work indicates that the predominant pathway taken by the HRP was via intercellular spaces and it is suggested that the permeability between junctional complexes of parenchymal cells is capable of being modified in vivo.

Animals↗
PubMed1977

Effects of secretory nerve stimulation on acid phosphatase and peroxidase in submandibular saliva and acini in cats.

The effects of parasympathetic or sympathetic nerve stimulation either alone, or in combination, on the acid phosphatase-containing central acinar cells and the peroxidase-containing demilunar cells of the cat submandibular salivary gland have been investigated by histochemical and cytochemical techniques. The results obtained with these techniques were correlated with biochemical assays for both enzymes in the saliva secreted. The results indicate that, although both sets of nerves probably affect both sets of cells, the predominant secretory effect of parasympathetic stimulation is on the central cells and, conversely, the predominant secretory effect of sympathetic stimulation is on the demilunes. Sympathetic stimulation appeared also to have initiated synthesis of peroxidase in the demilunar cells, especially when it was superimposed upon parasympathetic stimulation.

Acid Phosphatase↗
PubMed1977

Supporting effects of myoepithelial cells in submandibular glands of dogs when acting against increased intraluminal pressure.

1. In dogs under chloralose-urethane anaesthesia secretion from the submandibular gland was recorded with the outflow at gland level and at heights of up to 50 cm above the gland.2. With the outflow level increased, secretion elicited by sympathetic nerve stimulation was far better maintained before than after injection of the alpha-adrenoceptor blocking agent dihydroergotamine.3. When the outflow level was raised while no secretion occurred, fluid flowed into the gland. Part of this amount was returned on lowering the outflow to gland level. This volume was assumed to have been accommodated in the distended luminal system, whereas some fluid was obviously lost into the gland tissues.4. Both these fractions of the fluid flowing into the gland when the outflow level was high could be reduced by injecting the alpha-receptor agonist phenylephrine. Bradykinin, which like phenylephrine is known to contract salivary myoepithelial cells, had the same effect on the two inflow volumes.5. It is concluded that myoepithelial contraction reduced the distensibility of the luminal system and in addition supported the acini, thereby diminishing backflow into the glandular tissues and enabling the gland to discharge saliva against a high pressure.6. Morphologically it was found that in resting glands PAS-positive saliva was displaced from the ductal system when the outflow cannula was raised, but it was preserved in the lumina under similar conditions when the myoepithelial cells were being stimulated by phenylephrine or bradykinin.7. Although sympathetic secretion could be maintained against a head of pressure, so long as it was accompanied by myoepithelial contraction, the increased force caused by the secretion led to disruptive damage of striated ducts which are the first part of the luminal system not supported by myoepithelial cells.8. The morphological findings reinforce the belief that contraction of myoepithelial cells gives active support to the underlying parenchyma.

Animals↗
PubMed1977

Movement of horseradish peroxidase in submandibular glands of dogs after ductal injection.

Small volumes of solutions of a tracer enzyme, horseradish peroxidase (HRP), have been injected retrogradely via the main duct of submandibular glands in dogs, and the distribution of the peroxidase reaction product within the glands has been studied light microscopically and electron microscopically at different times after the injection. Tissue taken immediately after injections of 0.25 ml, given over 10 sec, showed that HRP had penetrated into the interstitial spaces of the gland. It was usually irregularly distributed and appeared to have reached the interstices of the gland by passing through the intercellular junctions of adjacent acinar cells. In time the tracer gradually disappeared from the interstices of the gland. Most of this removal was probably via lymphatic and blood vessels, into both of which peroxidase rapidly passed after ductal injection. At 5 hours after the HRP injection only very weak interstitial staining was present and at later times none was detected. An inflammatory reaction occurred in the gland, starting within minutes after the injection of this foreign protein and reaching a peak after about 12 hours. The events in dogs and those occurring in rabbits under similar experimental conditions have been compared. This study has given some indication how foreign substances may be distributed after retrograde injection into salivary glands.

Animals↗